Abstract 4508: Glypican-3 (GPC3) signaling pathway involved in breast cancer progression

Abstract

Abstract GPC3 is a proteoglycan downregulated in breast tumors. Employing LM3 murine mammary adenocarcinoma cell line (ER-, PR-), we have previously shown that GPC3 acts as a metastasis suppressor and induces a reversion of the epithelial to mesenchymal transition (EMT). GPC3 overexpressing LM3 cells presented an inhibition of canonical Wnt and Akt pathways. On the other hand, non-canonical Wnt (JNK) and p38 signaling were activated. However, the hierarchical sequence through which GPC3 modulates those pathways has not been determined yet.The aim of this work was to study the molecular mechanism involved in the GPC3 effect on breast tumor progression, focusing on canonical Wnt pathway. We investigated the receptor complex with which GPC3 is interacting and the relationships among the pathways modulated by this glypican. We determined by IP that GPC3 could physically interact with the Wnt receptor Fz. Our results suggested that GPC3 binds to Fz and blocks the binding of the canonical Wnt factors. We also confirmed by the determination of cytoplasmic β-Catenin levels through WB and their transcriptional activity employing gene reporter assay, that GPC3 is inhibiting this pathway. We showed by qPCR array that GPC3 also modulates canonical Wnt signaling in a genomic way. Of 84 examined genes, only Wnt5b (non-canonical factor) was upregulated in LM3-GPC3 cells, 66 genes were downregulated (several Fzds, LRP5/6, DVL1 and DVL2, Frat1, Ccnd1, Ccnd2, Ccnd3, Csnk1d, jun and myc, among others), and the expression of 17 genes was not modified (including several Wnt factors). Finally, LM3-GPC3 cells were treated with SB203589, SP600125 (p38 and JNK pharmacological inhibitors respectively), LiCl (canonical Wnt activator) or transduced with a constitutively active variant of Akt1 (CA Akt). The effect of the treatments on the other pathways activity was evaluated by WB. We showed that the Akt inhibition found in LM3-GPC3 cells is required for the activation of the non-canonical Wnt pathway, as well as for the inhibition of the canonical one, but it had no effects on p38 signaling. p38 activation was necessary for non-canonical Wnt upregulation and for Akt and canonical Wnt inhibition. Canonical Wnt inhibition was essential for Akt downregulation as well as for p38 and non-canonical Wnt signaling activation. We demonstrated that in addition to the significant role of JNK in the non-canonical Wnt pathway, it also regulates canonical signalling. Non-canonical Wnt activity also participates in p38 activation but has no effect on Akt activity.In sum, we have described the receptor complex with which GPC3 interacts, as well as the canonical Wnt signaling inhibition induced by GPC3 - both genomic and non-genomic - and finally several interactions among Wnt, Akt and p38 pathways. We believe that our results provide additional information regarding the molecular mechanism of GPC3 involved in metastasis suppression. Citation Format: Dolores Fernandez, Magali Cercato, Macarena Guereño, Maria G. Peters. Glypican-3 (GPC3) signaling pathway involved in breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4508.